The first Me 321 A-1 production aircraft entered service in May 1941 with Grossraumlastensegler 321 at Leipheim, initially towed by Ju 90s and later by the He 111Z and the Troikaschlepp arrangement of three Bf 110s. The later Me 321 B-1 variant had a crew of three and was armed with four 7.92 mm (.312 in) MG 15 machine guns. In spring 1942, the remaining Me 321s were withdrawn from service in Russia in anticipation of the planned Operation Herkules, the invasion of Malta, in which a fleet of the gliders hauled by He 111Zs were to be used. The plan was abandoned due to a lack of towing aircraft.

In 1943, the Me 321s were returned to Russia to be used in a projected operation to relieve General Friedrich Paulus' besieged army at Stalingrad, but by the time they reached the front line, no suitable airfields remained and they were sent back to Germany.

Following the cancellation of the Stalingrad operation, the Me 321 gliders were either mothballed or scrapped, though some were converted into the powered variant, the Me 323 with six 895 kW (1,200 hp) engines. This was the biggest land-based cargo aircraft of World War II. A further proposed operation — in which the remaining Me 321s would have landed troops on Sicily — was also abandoned, due to a lack of suitable landing sites. Ultimately, 200 Me 321s were produced.